Agents affecting thrombosis and hemostasis

ABSTRACT

Analogs of blood factors which are transiently inactive are useful in treatment of diseases characterized by thrombosis. In addition, modified forms of activated blood factors that generate the active blood factor in serum but have extended half-lives are useful in treating hemophilia conditions. These modified forms of the blood factor may be acylated forms which are slowly deacylated in vivo.

This application is a continuation of Ser. No. 08/469,301, filed Jun. 6,1995, now U.S. Pat. No. 5,837,679, which is a divisional application ofSer. No. 08/268,003, filed Jun. 29, 1994, now U.S. Pat. No. 5,583,107,which is a continuation-in-part of Ser. No. 08/249,777, filed May 26,1994, now U.S. Pat. No. 5,597,799, which is a continuation of Ser. No.07/808,329, filed Dec. 16, 1991, now abandoned, which is acontinuation-in-part of Ser. No. 07/578,646, filed Sep. 4, 1990 and nowU.S. Pat. No. 5,278,144.

TECHNICAL FIELD

The invention relates to peptide drugs for regulation of hemostatic andthrombotic processes. The invention also concerns coagulation factorswhose protease or enzymatic activity has been transiently inactivated.

BACKGROUND ART

Hemostasis, the control of bleeding, occurs by surgical means, or by thephysiological properties of vasoconstriction and coagulation. Thisinvention is particularly concerned with blood coagulation and ways inwhich it assists in maintaining the integrity of mammalian circulationafter injury, inflammation, disease, congenital defect, dysfunction orother disruption. After initiation of clotting, blood coagulationproceeds through the sequential activation of certain plasma proenzymesto their enzyme forms. These plasma glycoproteins, including Factor XII,Factor XI, Factor IX, Factor X, Factor VII, and prothrombin, arezymogens of serine proteases. Most of these blood clotting enzymes areeffective on a physiological scale only when assembled in complexes onmembrane surfaces with protein cofactors such as Factor VIII and FactorV. Other blood factors modulate and localize clot formation, or dissolveblood clots. Activated protein C is a specific enzyme that inactivatesprocoagulant components. Calcium ions are involved in many of thecomponent reactions. Blood coagulation follows either the intrinsicpathway, where all of the protein components are present in blood, orthe extrinsic pathway, where the cell-membrane protein tissue factorplays a critical role. Clot formation occurs when fibrinogen is cleavedby thrombin to form fibrin. Blood clots are composed of activatedplatelets and fibrin.

Thrombin is a multifunctional protease that regulates several keybiological processes. For example thrombin is among the most potent ofthe known platelet activators. In addition, as described above, thrombinis essential for the cleavage of fibrinogen to fibrin to initiate clotformation. These two elements are involved in normal hemostasis but inatherosclerotic arteries can initiate the formation of a thrombus, whichis a major factor in pathogenesis of vasoocclusive conditions such asmyocardial infarction, unstable angina, nonhemorrhagic stroke andreocclusion of coronary arteries after angioplasty or thrombolytictherapy. Thrombin is also a potent inducer of smooth cell proliferationand may therefore be involved in a variety of proliferative responsessuch as restenosis after angioplasty and graft induced atherosclerosis.In addition, thrombin is chemotactic for leukocytes and may thereforeplay a role in inflammation. (Hoover, R. J., et al. Cell (1978) 14:423;Etingin, O. R., et al., Cell (1990) 61:657.) These observations indicatethat inhibition of thrombin formation or inhibition of thrombin itselfmay be effective in preventing or treating thrombosis, limitingrestenosis and controlling inflammation.

The formation of thrombin is the result of the proteolytic cleavage ofits precursor prothrombin at the Arg-Thr linkage at positions 271-272and the Arg-Ile linkage at positions 320-321. This activation iscatalyzed by the prothrombinase complex, which is assembled on themembrane surfaces of platelets, monocytes, and endothelial cells. Thecomplex consists of Factor Xa (a serine protease), Factor Va (acofactor), calcium ions and the acidic phospholipid surface. Factor Xais the activated form of its precursor, Factor X, which is secreted bythe liver as a 58 kd precursor and is converted to the active form,Factor Xa, in both the extrinsic and intrinsic blood coagulationpathways. It is known that the circulating levels of Factor X, and ofthe precursor of Factor Va, Factor V, are on the order of 10⁻⁷ M. Therehas been no determination of the levels of the corresponding activeFactors Va and Xa.

The amino acid sequences and genes of most of the plasma proteinsinvolved in hemostasis of blood have been determined, including FactorVIIa, Factor IXa, Activated Protein C, Factor X and Factor Xa. FIG. 1shows the complete sequence of the precursor form of Factor X asdescribed by Davie, E. W., in Hemostasis and Thrombosis, Second Edition,R. W. Coleman et al. eds. (1987) p. 250. Factor X is a member of thecalcium ion binding, gamma carboxyglutamyl (Gla)-containing, vitamin Kdependent, blood coagulation glycoprotein family, which also includesFactors VII and IX, prothrombin, protein C and protein S (Furie, B., etal., Cell (1988) 53:505).

As shown in FIG. 1, the mature Factor X protein is preceded by a40-residue pre-pro leader sequence which is removed during intracellularprocessing and secretion. The mature Factor X precursor of Factor Xa isthen cleaved to the two-chain form by deletion of the three amino acidsRKR shown between the light chain C-terminus and activationpeptide/heavy chain N-terminus. Finally, the two chain Factor X isconverted to Factor Xa by deletion of the "activation peptide" sequenceshown at the upper right-hand portion of the figure (numbered 1-52),generating a light chain shown as residues 1-139, and a heavy chainshown as residues 1-254. These are linked through a single disulfidebond between position 128 of the light chain and position 108 of theheavy chain. As further indicated in the figure, the light chaincontains the Gla domain and a growth factor domain; the proteaseactivity resides in the heavy chain and involves the histidine atposition 42, the aspartic at position 88, and a serine at position 185,circled in the figure.

There are two known pathways for the activation of the two-chain FactorX in vivo. Activation must occur before the protease is incorporatedinto the prothrombinase complex (Steinberg, M., et al., in Hemostasisand Thrombosis, Coleman, R. W., et al. eds. (1987) J. B. Lippencott,Philadelphia, Pa., p. 112). In the intrinsic pathway, Factor X iscleaved to release the 52-amino acid activation peptide by the "tenase"complex which consists of Factor IXa, Factor VII and calcium ionsassembled on cell surfaces. In the extrinsic pathway, the cleavage iscatalyzed by Factor VIIa which is bound to a tissue factor on membranes.Also of interest herein is the ability to convert Factor X to Factor Xaby in vitro cleavage using a protease such as that contained inRussell's viper venom. This protease is described by DiScipio, R. G., etal., Biochemistry (1977) 6:5253.

In some circumstances, it is desirable to interfere with the functioningof Factor Xa in order to prevent excessive clotting. However in others,such as in hemophilia, it is desirable to provide a source of Factor Xaindependent of the activation process that takes place in normalindividuals. Both of the common forms of hemophilia involve deficienciesin only the intrinsic pathway of activation, but the operation of theextrinsic pathway does not appear to be successful in arrestingbleeding.

The most common forms of hemophilia are hemophilia A which reflects adeficiency in the functioning of Factor VII, and hemophilia B whichreflects a deficiency in the functioning of Factor IX (also known asChristmas factor). These forms of hemophilia are well known. Similarly,other patients are treated currently for deficiencies of other bloodfactors (VII, X, XI, XIII) or von Willebrand's disease. Factor VIIdeficiency is not as clinically well-defined as hemophilia A or B,however patients with Factor VII deficiency have been reported to haveextensive bleeding. Protein C deficiency is associated with thromboticrisk.

Currently hemophiliacs (and other individuals with factor deficiencies)are treated with clotting (or other blood) factors on a prophylacticbasis, however current treatment strategies are not entirelysatisfactory. It is known for example that large numbers of hemophiliapatients develop inhibitors to clotting factors, and these patients arethen treated with products known as "bypass factors", such as Factor VIIor Factor IX complexes, or activated Factor IX complexes, or factorsfrom other mammalian species, such as porcine Factor VII. In turn, somebypass factors have disadvantages, such as being thrombogenic(especially in immobile patients), or by lack of specificity. Even inthe same patient, it has been shown that these therapies can be reliableon one administration and not effective on another (Lusher, J. M.,Management of Hemophiliacs with Inhibitors, Hemophilia in the Child andAdult, M. Hilgartner and C. Pochedly, eds., New York, Raven Press,1989.).

There exists a need for improved treatments for hemophilia and otherblood factor deficiencies.

For hemophilia patients, since a deficiency in either of factors VIII orIX result in an inadequate supply of Factor Xa, provision of Factor Xashould be effective in treatment of both hemophilias. In addition, anumber of instances have been found wherein Factor X itself is incapableof providing an active Factor Xa. This relatively rare class ofcongenital disorders has been described, for example, by Reddy, S. B.,et al., Blood (1989) 74:1486-1490; Watzke, H. H., et al. J Biol Chem(1990) 2:11982-11989; Hassan, H. J., et al., Blood (1988) 71:1353-1356;Fair, D. S., et al., Blood (1989) 73:2108-2116; and by Bernardi, F., etal., Blood (1989) 73:2123-2127.

Factor Xa, and several other activated blood factors, have notheretofore been useful as pharmaceuticals because of their extremelyshort half-life in serum, which for example typically is only about 30seconds for Factor Xa. In the invention described below, the half-lifeof these agents in serum is extended by providing a transientlyinactivated, slow release form, preferably an acylated form. In certainembodiments relating to Factor X, an acyl group is bound to the serineat the active site and inhibits clearance and is only slowly hydrolyzedto generate the active form of Factor Xa. In similar fashion, thisinvention also relates to other transiently inactivated blood factors,including activated Protein C, Factor IXa and Factor VIIa.

The use of acylation to prolong the half-life of certain blood clottingfactors has been disclosed. For example, Cassels, R. et al. Biochem J(1987) 247:359-400 found that various acylating agents remained bound tourokinase, tPA and streptokinase-plasminogen activator complex for timeperiods ranging from a half-life of 40 minutes to a half-life of over1,000 minutes depending on the nature of the acylating group and thenature of the factor. Acylation of tPA or streptokinase is alsodisclosed in U.S. Pat. No. 4,337,244. The use of an amidinophenyl groupfunctioning as an arginine analog to introduce, temporarily, asubstituted benzoyl group into the active site for the purpose ofenhancing serum stability was discussed by Fears, R. et al., Seminars inThrombosis and Homeostasis (1989) 15:129-139. This more general reviewfollowed a short report by Fears, R. et al. in Drugs (1987) 33: Supp. 357-63. Sturzebecher, J. et al. also reported stabilized acyl derivativesof tPA in Thrombosis Res (1987) 47:699-703. An additional report of theuse of the acylated plasminogen streptokinase activator complex (APSAC)was published by Crabbe, S. J. et al. Pharmaco-therapy (1990)10:115-126. Acylated forms of thrombin have also been described.

Returning to the function of Factor Xa per se, the activity of Factor Xain effecting the conversion of prothrombin to thrombin is dependent onits inclusion in the prothrombinase complex. The formation of theprothrombinase complex (which is 278,000 fold faster in effecting theconversion of prothrombin to thrombin than Factor Xa in soluble form)has been studied (Nesheim, H. E., et al., J Biol Chem (1979) 254:10952).These studies have utilized the active site-specific inhibitor, dansylglutamyl glycyl arginyl (DEGR) chloromethyl ketone, which covalentlyattaches a fluorescent reporter group into Factor Xa. Factor Xa treatedwith this inhibitor lacks protease activity, but is incorporated intothe prothrombinase complex with an identical stoichiometry to that ofFactor Xa and has a dissociation constant of 2.7×10⁻⁶ M (Nesheim, M. E.,J Biol Chem (1981) 256:6537-6540; Skogen, W. F., et al., J Biol Chem(1984) 256:2306-2310; Krishnaswamy, S., et al., J Biol Chem (1988)263:3823-3824; Husten, E. J., et al., J Biol Chem (1987)262:12953-12961).

Known methods to inhibit the formation of the prothrombinase complexinclude treatment with heparin and heparin-like compounds. This resultsin inhibition of the formation of the complex by antithrombin III inassociation with the heparin. Other novel forms of Factor Xa inhibitioninclude lipoprotein-associated coagulation inhibitor (LACI) (Girard, T.J., et al., Nature (1989) 383:518; Girard, T. J., et al., Science (1990)248:1421), leech-derived antistatin (Donwiddie, C., et al., J Biol Chem(1989) 264:16694), and tick-derived TAP (Waxman, L., et al., Science(1990) 248:593). Alternatively, agents which inhibit the vitaminK-dependent Gla conversion enzyme, such as coumarin, have been used.None of these approaches have proved satisfactory due to lack ofspecificity, the large dosage required, toxic side effects, and the longdelay in effectiveness.

DISCLOSURE OF THE INVENTION

The invention provides effective therapeutic agents for the regulationof hemostasis, and for the prevention and treatment of thrombusformation and other pathological processes in the vasculature induced bythrombin such as restenosis and inflammation. This is highly significantas thrombus formation is the leading cause of death in Westernsocieties, and restenosis is an expanding problem with increased use ofangioplasty and other invasive procedures.

The therapeutic materials of the invention are inactive (eitherpermanently or transiently) forms of mammalian blood factors includingFactor IXa, Factor VIIa, activated Protein C, and Factor Xa.

Certain aspects of this invention relate to permanently inactive formsof Factor Xa which are nevertheless capable of incorporationprothrombinase complex, thus preventing the formation of activeprothrombinase complex from endogenous Factor Xa. These pharmaceuticalsare especially useful in acute settings to prevent thrombosis. Thisincludes preventing thrombus formation in the coronary arteries ofpatients with rest angina, preventing rethrombosis after thrombolysis,and prevention of thrombosis during complicated angioplasties. Thesepharmaceuticals will also be useful in preventing smooth muscle cellproliferation following angioplasty or other vascular invasiveprocedures. The invention therapeutics offer considerable advantage overthe now standard treatment which involves heparin (Hanson, R. S., etal., Proc Nat Acad Sci (1988) 85:3184). The compounds of certain aspectsof this invention are double- or single-chain polypeptides which arecapable of participation in the prothrombinase complex, but which resultin an inactive complex.

In one aspect, the invention is directed to a two-chain polypeptide,designated Factor Xai, which is capable of forming the prothrombinasecomplex, but which results in a complex that lacks proteolytic activity.This two-chain polypeptide may be formed from one of two types of novelprecursors. One type, designated herein Factor Xi, has substantially theamino acid sequence of Factor X, but is modified as described herein soas to result in an inactive two-chain polypeptide, Factor Xai, whencleaved by normal coagulation processing proteases or by in vitrotreatment with Factor X activator from viper venom. The other type,designated herein Factor X'i, is a truncated form of single chain FactorX wherein the proteolytic cleavage site (or portion or extensionthereof) at the C-terminus of the light chain, shown as RXR in FIG. 1,is ligated directly (with the optional addition of one or several aminoresidues) to the N-terminus of the activated form of the heavy chain asshown in one embodiment in FIG. 3. Upon cleavage, Factor X'i alsoresults in the two-chain Factor Xai of the invention which results in aprothrombinase complex lacking proteolytic activity. Of course, theactive cofactor, Factor Xa, could also be generated by using theanalogous precursors of the Factor X' type illustrated in FIG. 2.

Thus, in other aspects, the invention is directed to the Factor Xaitwo-chain prothrombinase complex, and to the novel precursors of theFactor Xai therapeutic proteins, to the DNA sequences encoding them, andto recombinant materials and methods generally which permit theirproduction.

Other aspects of the invention include pharmaceutical compositions ofthe therapeutically useful Factor Xai proteins and to methods to preventor treat thrombosis or other pathological events initiated by thrombinusing these compositions. In certain other aspects of this invention,transiently inactivated blood proteins such as activated Protein C areused as antithrombotics, where controlled, slow-release formulations aredesired.

This invention is also directed to transiently inactivated blood factorswhich are suitable for use as procoagulents, such as for wound healing,as bypass factors or in replacement therapy, or other treatments forhemophilia.

The availability of recombinantly produced and plasma derived bloodfactors provides a convenient source for these materials for use asprocoagulants, antithrombotics, or in the treatment of hemophilia. Forexample, in the practice of this invention, Factor Xa, whetherrecombinantly produced directly, obtained from recombinantly producedFactor X by activation using, for example, Russell's Viper Venom, orisolated from plasma and similarly converted to Factor Xa can beconverted to form a usable pharmaceutical by extending its half-life inserum. This can be accomplished by acylation of the serine at the activesite which provides a slow release form of the active factor, alsoreferred to herein as a "transiently inactivated" blood factor.Transient inactivation, via acylation or other means, of the other bloodfactors described in this invention confers similar slow-releasefeatures.

Thus, in certain embodiments, the invention is directed to Factor Xawherein the serine residue at position 185 of the heavy chain isacylated with an agent which permits its appropriately timed conversionto active Factor Xa. Other aspects of the invention includepharmaceutical compositions for the treatment of hemophilia containingacylated Factor Xa, Factor IXa, and Factor VIIa of this invention and tomethods to treat hemophilia using these compositions.

The transiently inactivated blood factors of this invention, theirderivatives, or their antibodies are formulated into physiologicallyacceptable vehicles, especially for therapeutic, imaging and otherdiagnostic use. Such vehicles include sustained-release formulations. Acomposition is also provided comprising a transiently inactivated bloodfactor and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of human Factor X and its relevant cleavagesites as described in the prior art.

FIG. 2 shows the structure of one embodiment of a single-chain Factor X'which is a precursor to yield a two-chain cleavage product that willparticipate in prothrombinase complex formation. The form shown in thisfigure will produce a two-chain peptide which retains proteolyticactivity in the complex; a modified form, as described below, iscatalytically inactive.

FIG. 3 shows one embodiment of Factor X'i.

FIG. 4 shows the cDNA sequence encoding Factor X.

FIGS. 5a and 5b show a Western blot of recombinantly produced,potentially active Factor X and Factor Xa.

FIG. 6 is a Western blot of recombinantly produced, inactivated forms ofFactor X and Factor Xa.

FIGS. 7a-7d consist of a series of Lineweaver-Burk plots showing theenzymatic activity of native and recombinantly produced Factor Xconverted to activated form.

FIGS. 8a-8d show a comparison of prothrombinase complex activity ofvarious Factor X forms.

FIG. 9 shows the result of a two-stage prothrombin clotting assay forvarious forms of Factor X.

FIG. 10 shows inhibition of prothrombinase complex formation by inactiveforms of Factor X.

FIG. 11 shows residual amidolytic activity of human factor Xa afteracylation.

FIGS. 12a and 12b shows the activation of acyl Xa.

FIG. 13 shows the effect of infusion of p-anisoyl factor Xa in rabbits.

FIG. 14 shows the activation of acyl activated protein C.

FIG. 15 shows the activation of o-anisoyl factor VIIa.

MODES OF CARRYING OUT THE INVENTION

In general, one aspect of the invention encompasses the therapeuticallyuseful two-chain polypeptide, designated Factor Xai herein, and thesingle-chain precursors of this two-chain protein. These peptides areabout 80% homologous, preferably about 90% homologous to the amino acidsequences shown at positions 1-139 (light chain) and 1-254 (heavy chain)in FIG. 1. It should be noted that in FIG. 1, the pre-pro leadersequence is numbered -40 through -1, prior to the numbering beginning atthe N-terminus of the light chain. The light chain is numbered 1-139.The intervening tripeptide RKR, which, in mature Factor X, is deleted,is not numbered. The activation peptide beginning subsequent to thisintervening tripeptide is numbered 1-52; the isoleucine referred tohereinbelow as "position 53" of the activation peptide is, in fact, thefirst amino acid of the heavy chain in the activated form. This restartsthe numbering shown in the figure, and the heavy chain is numbered1-254.

The embodiments of the two-chain peptide, Factor Xai, are effective informing the prothrombinase complex, as determined by their ability toinhibit (or compete with) the formation of the native prothrombinasecomplex involving Factor Xa. Their ability to inhibit prothrombinasecomplex formation can be determined conveniently by the method ofKrishnaswamy, S., J Biol Chem (1988) 263:3823-3834, cited above.However, when incorporated into the prothrombinase complex, the complexfails to show its proteolytic activity, as determined by the method ofvan Dieijen, G., et al., J Biol Chem (1981) 256:3433 or of Skogen, W. F.et al., J Biol Chem (1984) 256:2306. These Factor Xai proteins may ormay not be immunoreactive with antibodies against native Factor Xa oragainst Factor X, including commercially available antibodies specificfor human Factor X. The Factor Xai proteins are antithromboticmaterials.

The invention is also directed to precursors of the foregoing inactivecompetitors with Factor Xa. One group of these precursors are novelmodified forms of Factor X designated Factor X', wherein one or more ofthe residues at position 42, 88 or 185 of the heavy chain are convertedto alternate amino acid residues, thus inactivating the proteolyticproperties of the peptide. The modified forms of Factor X contain at aminimum the light chain sequence and the heavy chain sequence to whichis attached the activation peptide. The intervening tripeptide (betweenthe C-terminus of the light chain the N-terminus of the activationpeptide) and the pre-pro leader sequence may or may not be present.Thus, the Factor X may either be a single-chain protein when thetripeptide is included) or a two-chain precursor of Factor Xa (when thetripeptide has been deleted).

Preferably, the alteration at the residues of the protease active siteis either a deletion, or a conversion to a conservative, substitutedamino acid so as to maintain the three-dimensional conformation of thetwo-chain protein. By "conservative" is meant a substitution whichmaintains the correct conformation, rather than a substitution whichmaintains the correct activity. Thus, the histidine residue at position42 preferably replaced by phenylalanine; the aspartic acid at position88 is preferably replaced by asparagine or glutamine, and the serineresidue at position 185 is preferably replaced by alanine or glycine.

Another group of precursors to the antithrombotic dimeric peptides ofthe invention is designated Factor X'i. In Factor X'i precursors, atleast a substantial portion of the activation peptide, preferably theentire activation peptide, is deleted. The precursors to the two-chainform of Factor X'i, however, must retain a proteolytic cleavage sitebetween the light and heavy chains. Therefore, amino acids subject toendogenous proteolysis are conveniently included in a single-chainprecursor form which extends the carboxy terminus of the light chain byvirtue of the cleavage site to the N-terminus of the heavy chain. Atypical embodiment of the single-chain precursor (including the pre-proleader) to the two-chain Factor X'i, which will now automatically beactivated by virtue of the absence of the activation peptide sequence(thus, becoming a Factor Xai) is shown in FIG. 3. In this embodiment,the hexapeptide sequence RKRRKR connects the C-terminus of the lightchain directly to the isoleucine residue at the N-terminus of the heavychain. Cleavage of this single-chain Factor X'i results in X'ai.

In constructing such modified X'i type precursors, a hydrophobic aminoacid must be retained at the N-terminus of the heavy chain (nativelyisoleucine). See, for example, Dayhoff, M. O., "Atlas of ProteinSequence and Structure" (1972) 5:89-99 (Biomed. Res. Foundation, Wash.D.C.) and Greer, J., J. Molec. Biol. (1981) 153:1043-1053. It is evidentthat the single-chain Factor X' precursors are also novel and, whencleaved by proteolysis, yield Factor Xa, the normal enzymatically-activeform of the dimeric protein. This corresponding construction is shown inFIG. 2.

Thus, when the single-chain precursors of either Factor Xa or Factor Xaiare produced recombinantly in suitable host cells, the endogenousenzymes of the host cell may (1) cleave the single-chain precursorFactor X or X' to a two-chain form and, in the case of single-chainFactor X or X', further activate the factor by cleavage of theactivation peptide; in the case of Factor X' single-chain precursors,there is no activation peptide present, so the single-chain precursorwill automatically be activated when cleaved into a dimeric peptide. ForFactor X precursors, the double-chain form containing the activationpeptide may also be cleaved in vitro using a suitable protease, such asthe Factor X activator of Russell's viper venom. Either Factor Xa orFactor Xai will be obtained depending on whether the active site hasbeen inactivated by alteration at the appropriate codons as furtherdescribed hereinbelow.

To summarize the terminology used in this application, the followingglossary may be useful:

"Factor X" refers to the native or recombinantly produced single- ortwo-chain Factor X sequence, essentially as shown in FIG. 1, containingat a minimum the heavy chain to which is attached the activationpeptide, at its N-terminus, and the light chain. These may or may not belinked through a cleavage sequence as indicated in the figure.

"Factor IX", "Factor VII" and "activated Protein C" refer to therespective native or recombinantly produced protein sequence as commonlyknown.

"Blood factor" refers to blood coagulation factors generally, andpreferably to a group of blood factors including Factor X, Factor VII,Factor IX, and Protein C, in their inactive, active, or inactivatedactive forms.

"rX" refers specifically to the recombinantly produced form of thisfactor.

"Factor Xi" refers to the recombinantly produced form of Factor X whichlacks proteolytic activity by virtue of the modification of the act asdescribed above. The designation "rXi" is also used for this protein.

"Factor Xa" refers to native or recombinantly produced, enzymaticallyactive dimer containing light and heavy chain only. The activationpeptide is not present in this complex.

"rXa" refers specifically to this complex when produced recombinantly.

"Factor Xai" refers to the modified form of Factor Xa which is activatedin the sense that it combines to form the prothrombinase complex, butwhich has no serine protease activity by virtue of the modification ofits active site. As this protein is produced only by recombinantmethods, "IrXai" is also used to designate this complex.

"Factor X'" refers to a modified, single-chain form of Factor X whichincludes only the light chain, heavy chain, and an intermediate,specific proteolytic cleavage site, such as that shown in FIG. 2. Thissingle-chain precursor may also contain the pre-pro sequence. As it is aresult only of recombinant production, it is also designated "rX'." Whencleaved by a protease so as to become activated, the products areindistinguishable from Factor Xa (or rXa) and, accordingly, thisterminology is again used.

Similarly, "Factor X'i" refers to a modified form of Factor X' which hasbeen inactivated at its catalytic site as described above. One form isshown in FIG. 3. Upon conversion to the two-chain form, as theactivation peptide is not present in the precursor, the products areindistinguishable from Factor Xai or rXai.

"Acylated Factor Xa" or "AcXa", unless otherwise specified, refers toFactor Xa, whether produced recombinantly or not, wherein the serineresidue at position 185 has been blocked with a substituent whichprovides the Factor Xa with a half-life in serum of at least 5-10minutes, preferably more than 15 minutes, and which releases Factor Xain active form over this time period. The half-life in serum can bemeasured directly in vivo using a suitably labeled form. However, it ispreferable to assess the ability of the extended life AcXa to generatethe active factor within the required time frame in vitro using as acriterion in vitro assays for which Xa is a catalyst. Under theseconditions, suitable forms of AcXa for the invention include those whichhave a rate constant for hydrolysis in isotonic aqueous media at pH 7.4and 37° C. such that a half-life of approximately 5 minutes to severalhours is achieved. The half-life can be determined directly in vitro bymeasuring the rate of hydrolysis of the acylated Xa, if desired, usingits ability to activate clotting, or the prothrombinase reaction ascriteria for Xa formation.

The blood factors described in this invention are defined herein to beany isolated polypeptide sequence which possesses a biological propertyof the naturally occurring blood factor polypeptide comprising acommonly known polypeptide sequence, variants and homologues thereof,and mammalian or other animal analogues.

"Biological property" for the purposes herein means an in vivo effectoror antigenic function or activity that is directly or indirectlyperformed by a blood factor (whether in its native or denaturedconformation), or by any subsequence thereof. Effector functions includereceptor binding, any enzyme activity or enzyme modulatory activity, anycarrier binding activity, any hormonal activity, any activity inpromoting or inhibiting adhesion of cells to an extracellular matrix orcell surface molecules, or any structural role. However, effectorfunctions do not include antigenic functions, i.e. possession of anepitope or antigenic site that is capable of cross-reacting withantibodies raised against a naturally occurring blood factorpolypeptide.

Ordinarily, the blood factors claimed herein will have an amino acidsequence having at least 75% amino acid sequence identity with acommonly known sequence, more preferably at least 80%, even morepreferably at least 90%, and most preferably at least 95%. Identity orhomology with respect to a commonly known blood factor sequence isdefined herein as the percentage of amino acid residues in the candidatesequence that are identical with the known blood factor amino acidresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology, and not consideringany conservative substitutions as part of the sequence identity. None ofN-terminal, C-terminal or internal extensions, deletions, or insertionsinto the blood factor sequence shall be construed as affecting homology.

Thus, the claimed transiently inactivated blood factor polypeptides andblood factors with extended plasma half-lives that are the subject ofthis invention include each blood factor sequence; fragments thereofhaving a consecutive sequence of at least 5, 10, 15, 20, 25, 30 or 40amino acid residues from a commonly known blood factor sequence; aminoacid sequence variants of a commonly known blood factor sequence whereinan amino acid residue has been inserted N- or C-terminal to, or within,the blood factor sequence or its fragment as defined above; amino acidsequence variants of the commonly known blood factor sequence or itsfragment as defined above has been substituted by another residue. Bloodfactor polypeptides include those containing predetermined mutations by,e.g., site-directed or PCR mutagenesis, and other animal species ofblood factor polypeptides such as rabbit, rat, porcine, non-humanprimate, equine, murine, and ovine blood factors, and alleles or othernaturally occurring variants of the foregoing and human sequences;derivatives of the commonly known blood factor or its fragments asdefined above wherein the blood factor or its fragments have beencovalently modified by substitution, chemical, enzymatic, or otherappropriate means with a moiety other than a naturally occurring aminoacid (for example a detectable moiety such as an enzyme orradioisotope); glycosylation variants of the blood factor (insertion ofa glycosylation site or deletion of any glycosylation site by deletion,insertion or substitution of appropriate amino acid); and soluble formsof the blood factor. Such fragments and variants exclude any transientlyinactivated blood factor polypeptide heretofore identified, includingany known protein or polypeptide of any animal species, which isotherwise anticipatory under 35 U.S.C. 102 as well as polypeptidesobvious over such known protein or polypeptides under 35 U.S.C. 103,including acylated thrombin, acylated tissue plasminogen factor,urokinase, and streptokinase.

Preparation of the Invention Peptides

The genomic organization and coding sequence for human Factor X areknown and the cDNA has been retrieved and sequenced (Leytus, S. P., etal., ProcNatl Acad Sci USA (1984) 81:3699; Kaul, R. K., et al., Gene1(1986) 41:311-314). The complete Factor X cDNA sequence is shown inFIG. 4. Full length sequences for other blood factors such as thrombin,Factors IXa and VIIa, and activated Protein C are well known in thefield. Throughout this specification, techniques described in relationto Factor X-related polypeptides are fully applicable to the other bloodfactors claimed in this invention, and are provided for exemplarypurposes only.

Full-length Factor X cDNA inserts are subcloned into M13mp18 or M13mp19vectors for site-directed mutagenesis. (The correct sequence encodingFactor X is verified by dideoxy sequencing.) Standard modificationtechniques are now readily available in the art, and thus the sequenceencoding Factor X is modified to obtain the DNA-encoding Factor X',Factor Xi, and Factor X'i.

The modified coding sequences for Factor X', Factor Xi, Factor X'i andthe other claimed blood factors are then ligated into suitableexpression vectors for recombinant production of the polypeptides. Inthe expression vectors, the prepro leader sequence is preferablyretained for expression in compatible host cells such as mammalianhosts. If bacterial or yeast expression is desired, it may be desirableto substitute a compatible leader sequence, such as the penicillinasesequence in bacteria, or the alpha-factor sequence in yeast.Alternatively, an ATG start codon may be directly placed before aminoacid 1 of the light chain-encoding sequence to produce an intracellularprotein.

The choice of host and expression control system is governed by thenature of the desired result. If endogenous activation by proteolyticcleavage is desired, mammalian systems may be preferable. However,production in microorganisms which provide simplicity of culturing isnot precluded, provided an in vitro system for carboxylation to producethe required carboxy glutamyl residue is employed, or the microorganismor other host natively lacking this posttranslational processing systemis transformed to provide it. A wide variety of expression systems forrecombinant DNA sequences is known in the art.

The modified DNA encoding Factor X', Factor Xi, Factor X'i or otherblood factor is preferably provided with linkers for ligation intocloning and expression vectors. Techniques for preparation of suchvectors are well understood in the art. The DNA encoding the desiredblood factor is ligated in operable linkage with control sequences,including promoters, upstream enhancers, termination sequences, and soforth, depending on the nature of the intended recombinant host cells.Technology is currently available for expression of heterologous genesin a variety of hosts, including prokaryotic hosts and variouseucaryotes, including yeast, mammalian or avian or insect cells, andplant cells. The choice of control sequences and markers in theexpression vectors is selected appropriately to these hosts.

For example, in prokaryotic hosts, various promoters, includinginducible promoters such as promoter and lambda phage P_(L) promoter canbe employed. Hybrid promoters such as the tac promoter, which containsthe trp polymerase binding region in combination with the lac operator,can be used. Suitable markers are generally those related to antibioticresistance. On the other hand, in mammalian cell cultures, commonly usedpromoters are virally derived, such as the early and late SV40 promotersand adenovirus promoters. Mammalian regulatable promoters, such as themetallothionein-II promoter may also be used. The metallothionein-IIpromoter is regulated by glucocorticoids or heavy metals. These promotersystems are compatible with typical mammalian hosts, the most commonlyused of which is Chinese hamster ovary (CHO) cells.

Another commonly employed system is the baculovirus expression systemcompatible with insect cells. Plant cells, used in conjunction with, forexample, the nopaline synthetase promoter, and yeast cells, used inconjunction with promoters associated with enzymes important in theglycolytic pathway, can also be employed. A number of suitableexpression systems can be found in appropriate chapters in "CurrentProtocols in Molecular Biology," Ausubel, F. M., et al., eds., publishedby Wiley Interscience, latest edition.

Certain preferred aspects of this invention relate to transientlyinactivated blood factors, such as Factors VIIa, IXa, Xa, and activatedProtein C. Transient inactivation may be accomplished by a variety ofmethods, including binding of an antibody/antibody fragment to theactive region, binding of moiety which blocks sterically the proteolyticor other active domain, or incorporation of a chemical moiety whichblocks the active blood factor domain and gradually is released from theblood factor. Particularly preferred embodiments of this invention areblood factor polypeptides which are transiently inactivated by beingacylated.

For purposes of this application, reversible inactivation of the bloodfactors of this invention may be accomplished using benzamidines, whichare good reversible inhibitors of trypsin-like enzymes. The cationicamidino group of the inhibitor interacts with an enzyme carboxylatelocated at the bottom of the S1 subsite. A wide variety of substitutedbenzamidines have been investigated as inhibitors of thrombin andplasmin and are suitable for practice of this invention (see e.g.,Andrews J M, Roman D P, Bing D M and Corey M. J Med Chem 21, 1202-1207,1978). Extensive studies have been reported on compounds containing twobenzamidine moieties, which are also desirable for the practice of thisinvention (see e.g., Tidwell R R, Webster W P, Shaver S R and Geratz JD. Thrombosis Research, 19, 339-349, 1980). Also useful for thisinvention is 1,2-bis(5-amidino 2-benzofuranyl) ethane, which is known toinhibit factor Xa with a Ki of 570 nM.

Also suitable for transient inactivation of proteins according to thisinvention are Kunitz inhibitors (a class of widely studied proteaseinhibitors). Bovine pancreatic trypsin inhibitor (aprotinin) and tissuefactor pathway inhibitor (also known as LACI) belong to this class.Dissociation constants (T_(1/2)) can range from 17 weeks to 11 seconds(Gebhard W, Tschesche H and Fritz H. Proteinase Inhibitors, Elsevier,1986). Aprotinin competitively inhibits factor VIIa with a Ki of 3 uM(Chabbat J, Porte P, Tellier M and Steinbuch M. Thrombosis Research, 71,205-215, 1993).

The acylated polypeptides of this invention, such as AcXa, AcIXa,AcVIIa, and Acylated activated Protein C, are prepared by standardacylation reaction of the corresponding blood factor, whetherrecombinantly produced or isolated from plasma, according to proceduresanalogous to those set forth, for example, or referenced in Cassels, R.et al. Biochem J (1987) 247:395-400 or U.S. Pat. No. 4,337,244 citedabove.

In certain embodiments of this invention, the blood factor is treatedwith a three to ten-fold molar excess of an acylating agent in a neutralpH buffer at room temperature. Catalytic activity is followed over atime course of approximately one to sixty, and preferably for ten tothirty minutes to assure the desired level of inactivation of protein.The reagent is preferable prepared as a 0.1M solution in DMSO and addedto the protein at pH 7.5. Blocked protein is subjected to gel filtration(preferably on a Sephadex G-25 column) at pH 5.0 to remove excessreagent. Protein may be stored at pH 5.0 at -70° C.-80° C. prior tofurther use.

Suitable active site acyl groups for use in this invention includebenzoyl, p or o methyl (toluoyl), p or o methoxy (p is a more preferredanisoyl), p or o fluoro benzoyl, Dimethyl acryloyl (3,3 or 3,4),Difluoro compounds, CH₃ CO benzene (acetyl gp), CH₃ CONH benzene(acetanilide), p or o ethoxy (or other alkyl groups), and guanidinobenzoyl.

Suitable esters for use in this invention include the 4-toluoyl ester,the 3,3-dimethyl acrylyl ester, cyclohexylidineacetyl ester, thecyclohex-1-enecarbonyl ester, the 1-methylcyclohexylidineacetyl ester,the 4-aminobenzoyl ester, the guanidinobenzoyl ester, the 4-anisoylester, the 4-N, N dimethylaminobenzoyl ester, and the PDAEB(4-N-(2-N'-(3-(2-pyridyldithio)-propenyl)amino-ethyl)aminobenzoyl ester.In general, the acylating agent will be the activated form of anon-toxic acid which provides a saturated, unsaturated or aromatic 5- or6-carbon ring to which a carboxyl is substituted. The ring may containfurther substitutions, such as amino, alkoxy, alkyl, additional ringsystems, or any other non-interfering non-toxic substituent. For FactorX and other blood factors with a catalytically active serine domain, anycompound capable of acylating the serine hydroxyl group or otherwiseblocking the serine catalytic domain in a reversible manner is suitablefor synthesis of the acylated blood factor. As described in U.S. Pat.No. 4,337,244, in general, either direct or inverse acylating agents canbe used. For direct acylating agents, the acylating moiety is itselfattracted to the catalytic site of the Factor Xa or other blood factor;in the inverse acylating approach, the leaving group is thus attracted.The acylated form of the blood factor is then purified from the reactionmixture using standard purification techniques, including dialysis,chromatography, selective extraction, and the like.

Potent acylating agents such as 3-alkoxy 4-chloroisocoumarins have beenreported for a variety of serine proteases (Harper J W and Powers J C.JACS 106, 7618-7619, 1984. Harper J W and Powers J C. Biochemistry 24,7200-7213, 1985) and are suitable for use in accordance with thisapplication. The stability of the acyl enzymes are dependent on thealkoxy groups, small groups give transiently stable (T_(1/2) <2 h) acylenzymes.

The compounds of the invention which serve as acylated blood factordiagnostics and/or pharmaceuticals must have an appropriate deacylationrate which assures an appropriate clearance time in vivo. The acylatedproteins reactivate in a time, temperature and pH dependent manner.Typically, deacylation is faster at 37° C. than at room temperature, andis faster at pH 8.0 than at pH 7.5. The deacylation rate can be measuredas having a half life of at least 5 minutes in vitro in buffer usingprothrombinase and/or clotting assays. Deacylation can be measureddirectly as described by Smith, R. A. G., et al., "Progress inFibrinolysis" (1985) Vol. VII, pp. 227-231 (Churchill Livingstone).Prothrombinase and clotting assays are described by Wolf, D. L., et al.J Biol Chem (1991) 266:13726.

In certain preferred embodiments, deacylation of acyl factors Xa and aPCis carried out by incubation in a solution of appropriate pH andassaying aliquots in an amidolytic or clotting assay. The relativeactivity is calculated as a percentage of equivalent amount of activefactor Xa or aPC carried through the same incubations. The preferredassay for acyl factor VIIa involves multiple steps. The acyl enzyme isincubated in the appropriate buffer at a protein concentration of 160nM. At each time point an aliquot is diluted to 0.16 nM and incubatedwith lipidated tissue factor (0.25 nM) for 1 min at room temperature.The factor VIIa/Tissue Factor mixture is then used for activation offactor X and resulting factor Xa assayed in an amidolytic assay.

Covalent modifications of the preferably acylated blood factors areincluded within the scope of this invention. Both native blood factorand amino acid sequence variants of the blood factor optionally arecovalently modified. One type of covalent modification included withinthe scope of this invention is a blood polypeptide fragment. Bloodfactor fragments having up to about 40 amino acid residues areconveniently prepared by chemical synthesis, or by enzymatic or chemicalcleavage of the full-length blood factor polypeptide or blood factorvariant polypeptide. Other types of covalent modifications of the bloodfactor or fragments thereof are introduced into the molecule by reactingtargeted amino acid residues of the blood factor or fragments thereofwith an organic derivatizing agent that is capable of reacting withselected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with a-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,a-bromo-b-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing a-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵ I or ¹³¹ I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R'--N═C═N--R'), where R and R' aredifferent alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking thetransiently inactivated blood factor to a water-insoluble support matrixor surface for use in the method for purifying anti-blood factorantibodies, and vice versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

The transiently inactivated blood factor of this invention optionally isfused with a heterologous polypeptide. The heterologous polypeptideoptionally is an anchor sequence such as that found in the decayaccelerating system (DAF); a toxin such as ricin, pseudomonas exotoxin,gelonin, or other polypeptide that will result in target cell death.These heterologous polypeptides are covalently coupled to the bloodfactor polypeptide through side chains or through the terminal residues.Similarly, other molecules toxic or inhibitory to a target mammaliancell (e.g. cancer cell) are coupled to the blood factor such astricothecenes, or antisense DNA that blocks expression of criticalgenes.

The transiently inactivated blood factor of this invention is covalentlymodified by altering its native glycosylation pattern. One or morecarbohydrate substituents in these embodiments are modified by adding,removing or varying the monosaccharide components at a given site, or bymodifying residues in the blood factor as glycosylation sites are addedor deleted.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Glycosylation sites are added to the blood factor of this invention byaltering its amino acid sequence to contain one or more of theabove-described tri-peptide sequences (for N-linked glycosylationsites). The alteration may also be made by the addition of, orsubstitution by, one or more serine or threonine residues to the bloodfactor (for O-linked glycosylation sites). For ease, the blood factor ispreferably altered through changes at the DNA level, particularly bymutating the DNA encoding the blood factor at preselected bases suchthat codons are generated that will translate into the desired aminoacids.

Chemical or enzymatic coupling of glycosides to the blood factorincreases the number of carbohydrate substituents. These procedures areadvantageous in that they do not require production of the polypeptidein a host cell that is capable of N- and O-linked glycosylation.Depending on the coupling mode used, the sugar(s) may be attached to (a)arginine and histidine, (b) free carboxyl groups, (c) free sulfhydrylgroups such as those of cysteine, (d) free hydroxyl groups such as thoseof serine, threonine, or hydroxyproline, (e) aromatic residues such asthose of phenylalanine, tyrosine, or tryptophan, or (f) the amide groupof glutamine. These methods are described in WO 87/05330, published Sep.11, 1987, and in Aplin and Wriston (CRC Crit. Rev. Biochem., pp. 259-306[1981]).

Carbohydrate moieties present on the blood factor also are removedchemically or enzymatically. Chemical deglycosylation requires exposureof the polypeptide to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the polypeptide intact. Chemicaldeglycosylation is described by Hakimuddin et al. (Arch. Biochem.Biophys., 259:52 [1987]) and by Edge et al. (Anal. Biochem., 118:131[1981]). Carbohydrate moieties are removed from the blood factor by avariety of endo- and exo-glycosidases as described by Thotakura et al.(Meth. Enzymol., 138:350 [1987]).

Glycosylation also is suppressed by tunicamycin as described by Duskinet al. (J. Biol. Chem., 257:3105 [1982]). Tunicamycin blocks theformation of protein-N-glycoside linkages.

The blood factor also is modified by linking it to variousnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

One preferred way to increase the in vivo circulating half life of acirculating blood factor is to conjugate it to a polymer that confersextended half-life, such as polyethylene glycol (PEG). (Maxfield, et al,Polymer 16,505-509 [1975]; Bailey, F. E., et al, in Nonionic Surfactants[Schick, M. J., ed.] pp. 794-821, 1967); (Abuchowski, A. et al., J.Biol. Chem. 252, 3582-3586, 1977; Abuchowski, A. et al, Cancer Biochem.Biophys. 7, 175-186, 1984); (Katre, N. V. et al., Proc. Natl. Acad.Sci., 84, 1487-1491, 1987; Goodson, R. et al. Bio Technology, 8,343-346, 1990). Conjugation to PEG also has been reported to havereduced immunogenicity and toxicity (Abuchowski, A. et al., J. Biol.Chem., 252, 3578-3581, 1977).

The blood factor also is entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-[methylmethacylate] microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, phospholipid vesicles, albuminmicrospheres, microemulsions, nano-particles and nanocapsules), or inmacroemulsions. Such techniques are disclosed in Remington'sPharmaceutical Sciences, 16th edition, Osol, A., Ed., (1980).

The transiently inactivated blood factors of this invention is alsouseful in generating antibodies, as standards in assays for the bloodfactor (e.g., by labeling the blood factor for use as a standard in aradioimmunoassay, enzyme-linked immunoassay, or radioreceptor assay), inaffinity purification techniques, and in competitive-type receptorbinding assays when labeled with radioiodine, enzymes, fluorophores,spin labels, and the like.

Those skilled in the art will be capable of screening variants in orderto select the optimal variant for the purpose intended. For example, achange in the immunological character of the transiently inactivatedblood factor, such as a change in affinity for a given antibody or forthe factor's natural receptor or ligand, is measured by acompetitive-type immunoassay using a standard or control such as anative blood factor. Other potential modifications of protein orpolypeptide properties such as redox or thermal stability,hydrophobicity, susceptibility to proteolytic degradation, stability inrecombinant cell culture or in plasma, or the tendency to aggregate withcarriers or into multimers are assayed by methods well known in the art.

Administration and Use

The Factor Xai peptides of the invention are prothrombinase inhibitorsand are thus useful in procedures complicated by thrombosis and inconditions whose pathogenesis involves thrombin generation. Theseconditions include those involving arterial thrombosis, such as unstable(i.e., rest) angina and abrupt vessel closure during vascularinterventions including coronary and peripheral angioplasty andatherectomy, and during and after vascular bypass procedures (peripheraland coronary), reocclusion after thrombolytic therapy for myocardialinfarction, thrombotic stroke (stroke in evolution), and thrombosis dueto vasculitis (Kawasaki's disease). Also included are conditionsinvolving venous thrombosis, such as deep venous thrombosis of the lowerextremities, pulmonary embolism, renal vein, hepatic vein, inferior venacava thrombosis, and cavernous sinus thrombosis. Other target conditionsare those involving diffuse activation of the coagulation system, suchas sepsis with disseminated intravascular coagulation, disseminatedintravascular coagulation in other settings, thrombotic thrombocytopenicpurpura, and rare conditions of unknown etiology (Lupus anticoagulant).

The Factor Xai of the invention is also useful as an anticoagulant andanti-inflammatory for cardiopulmonary bypass, in harvesting organs, inpreparation of blood products or samples and in transport andimplantation of organs and associated treatment of the recipient. TheFactor Xai, in a slow release form, is especially useful in indwellingintravascular devices (i.v.s, catheters, grafts, patches).

Thrombosis also plays. a role in restenosis following vascularinterventions such as angioplasty, atherectomy, or endarterectomy bydirectly or indirectly causing smooth muscle cell proliferation, and theFactor Xai of the invention is also useful in treating this condition.

Adult respiratory distress syndrome (ARDS) is thought to be an"endotoxin" disease in which a prothrombotic endothelium is likely toexist, with inflammatory and proliferative components; Factor Xai isalso useful in treatment of ARDS.

The transiently inactivated activated Protein C polypeptides of thisinvention are useful as antithrombotics. The transiently inactivatedFactors IXa, Xa, and VIIa of this invention are useful hemostaticfactors, particularly for the treatment of hemophilia as replacement orbypass factors. The modified blood factors of this invention, modifiedto extend their half-life in vivo, are useful in treating hemophiliawhether the origin of the hemophilia resides in the Factor X, IX, or VIIgene, or the more widespread types, hemophilias A and B.

Therapeutic formulations of the blood factors of this invention, or of ablood factor antibody are prepared for storage by mixing the bloodfactor polypeptide or antibody having the desired degree of purity withoptional physiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, supra), in the form of lyophilizedcake or aqueous solutions. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counter ions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

The blood factor or blood factor antibody to be used for in vivoadministration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes, and may be performedprior to or following lyophilization and reconstitution. The bloodfactor or antibody to the blood factor ordinarily will be stored inlyophilized form or in solution.

Therapeutic blood factor compositions, or blood-factor specific antibodycompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The blood factor, its antibody or variant may be optionally combinedwith or administered in concert with other agents known for use in thetreatment of particular coagulation disorders, such as thrombolytics(including tPA, streptokinase and urokinase), heparin, aspirin, Hirudin,Hirulog. When the blood factor is used to stimulate coagulation, it maybe combined with or administered in concert with other compositions thatstimulate coagulation.

The route of the blood factor or blood factor antibody administration isin accord with known methods, e.g., injection or infusion byintravenous, intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, or intralesional routes, or by sustained release systemsas noted below. The blood factor is preferably administered continuouslyby infusion or by bolus injection. Blood factor antibody is administeredin the same fashion, or by administration into the blood stream orlymph.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g. films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels [e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15: 167-277 [1981]and Langer, Chem. Tech., 12: 98-105 [1982] or poly(vinylalcohol)],polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 [1983]), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyricacid (EP 133,988). While polymers such as ethylene-vinyl acetate andlactic acid-glycolic acid enable release of molecules for over 100 days,certain hydrogels release proteins for shorter time periods. Whenencapsulated proteins remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for proteinstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S--S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Sustained-release blood factor or antibody compositions also includeliposomally entrapped blood factor or antibody. Liposomes containing theclaimed blood factor or antibody are prepared by methods known per se:DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980);EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patentapplication 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP102,324. Ordinarily the liposomes are of the small (about 200-800Angstroms) unilamelar type in which the lipid content is greater thanabout 30 mol. % cholesterol, the selected proportion being adjusted forthe optimal blood factor therapy. Liposomes with enhanced circulationtime are disclosed in U.S. Pat. No. 5,013,556. Additionally, Giles, A.R., et al. Brit J Hematol (1988) 69:491-497 describe the formulation ofFactor Xa in phosphatidylcholine-phosphatidylserine vesicles.

Another use of the present invention comprises incorporating the bloodfactor polypeptide or antibody into formed articles. Examples of sucharticles include vascular stents, grafts, surgical tubing, etc. Sucharticles can be used in modulating cellular growth and development. Inaddition, cell growth and division, and tumor invasion may be modulatedwith these articles.

An effective amount of the blood factor or antibody to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 1 mg/kg to up to 100 mg per patient, and more preferably of 1-50mg per patient per continuous injected dose, depending on the factorsmentioned above. Typically, the clinician will administer the bloodfactor or antibody until a dosage is reached that achieves the desiredeffect. The progress of this therapy is easily monitored by conventionalassays.

Certain aspects of this invention are directed to antibodies to theblood factors. The antibodies of this invention are obtained by routinescreening. Polyclonal antibodies to the blood factor generally areraised in animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of the blood factor and an adjuvant. It may be useful toconjugate the blood factor or blood factor fragment containing thetarget amino acid sequence to a protein that is immunogenic in thespecies to be immunized, e.g., keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctionalor derivatizing agent, for example, maleimidobenzoyl sulfosuccinimideester (conjugation through cysteine residues), N-hydroxysuccinimide(through lysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹ N═C═NR, where R and R¹ are different alkyl groups.

The route and schedule of immunizing an animal or removing and culturingantibody-producing cells are generally in keeping with established andconventional techniques for antibody stimulation and production. Whilemice are frequently immunized, it is contemplated that any mammaliansubject including human subjects or antibody-producing cells obtainedtherefrom can be immunized to generate antibody producing cells.

Subjects are typically immunized against the blood factor or itsimmunogenic conjugates or derivatives by combining 1 mg or 1 mg of bloodfactor immunogen (for rabbits or mice, respectively) with 3 volumes ofFreund's complete adjuvant and injecting the solution intradermally atmultiple sites. One month later the subjects are boosted with 1/5 to1/10 the original amount of immunogen in Freund's complete adjuvant (orother suitable adjuvant) by subcutaneous injection at multiple sites. 7to 14 days later animals are bled and the serum is assayed foranti-blood factor antibody titer. Subjects are boosted until the titerplateaus. Preferably, the subject is boosted with a conjugate of thesame blood factor, but conjugated to a different protein and/or througha different cross-linking agent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are used to enhance the immune response.

After immunization, monoclonal antibodies are prepared by recoveringimmune lymphoid cells--typically spleen cells or lymphocytes from lymphnode tissue--from immunized animals and immortalizing the cells inconventional fashion, e.g., by fusion with myeloma cells or byEpstein-Barr (EB)-virus transformation and screening for clonesexpressing the desired antibody. The hybridoma technique describedoriginally by Kohler and Milstein, Eur. J. Immunol. 6:511 (1976) hasbeen widely applied to produce hybrid cell lines that secrete highlevels of monoclonal antibodies against many specific antigens.

It is possible to fuse cells of one species with another. However, it ispreferable that the source of the immunized antibody producing cells andthe myeloma be from the same species.

Hybridoma cell lines producing anti-blood factor are identified byscreening the culture supernatants for antibody which binds to the bloodfactor. This is routinely accomplished by conventional immunoassaysusing blood factor preparations or by FACS using cell-bound blood factorand labeled candidate antibody.

The hybrid cell lines can be maintained in culture in vitro in cellculture media. The cell lines of this invention can be selected and/ormaintained in a composition comprising the continuous cell line inhypoxanthine-aminopterin thymidine (HAT) medium. In fact, once thehybridoma cell line is established, it can be maintained on a variety ofnutritionally adequate media. Moreover, the hybrid cell lines can bestored and preserved in any number of conventional ways, includingfreezing and storage under liquid nitrogen. Frozen cell lines can berevived and cultured indefinitely with resumed synthesis and secretionof monoclonal antibody. The secreted antibody is recovered from tissueculture supernatant by conventional methods such as precipitation, ionexchange chromatography, affinity chromatography, or the like. Theantibodies described herein are also recovered from hybridoma cellcultures by conventional methods for purification of IgG or IgM as thecase may be that heretofore have been used to purify theseimmunoglobulins from pooled plasma, e.g., ethanol or polyethylene glycolprecipitation procedures. The purified antibodies are sterile filtered,and optionally are conjugated to a detectable marker such as an enzymeor spin label for use in diagnostic assays of the blood factor in testsamples.

While mouse monoclonal antibodies routinely are used, the invention isnot so limited; in fact, human antibodies may be used and may prove tobe preferable. Such antibodies can be obtained by using human hybridomas(Cote et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985)). Chimeric antibodies, Cabilly et al., U.S. Pat. No.4,816,567, (Morrison et al., Proc. Natl. Acad. Sci., 81:6851 (1984);Neuberger et al., Nature 312:604 (1984); Takeda et al., Nature 314:452(1985)) containing a murine anti-blood factor variable region and ahuman constant region of appropriate biological activity (such asability to activate human complement and mediate ADCC) are within thescope of this invention, as are humanized anti-blood factor antibodiesproduced by conventional CDR-grafting methods.

Techniques for creating recombinant DNA versions of the antigen-bindingregions of antibody molecules (known as Fab or variable regionsfragments) which bypass the generation of monoclonal antibodies areencompassed within the practice of this invention. One extractsantibody-specific messenger RNA molecules from immune system cells takenfrom an immunized subject, transcribes these into complementary DNA(cDNA), and clones the cDNA into a bacterial expression system andselects for the desired binding characteristic. The Scripps/Stratagenemethod uses a bacteriophage lambda vector system containing a leadersequence that causes the expressed Fab protein to migrate to theperiplasmic space (between the bacterial cell membrane and the cellwall) or to be secreted. One can rapidly generate and screen greatnumbers of functional Fab fragments to identify those which bind theblood factor with the desired characteristics.

Antibodies capable of specifically binding to the proteolytically activedomains of the blood factors are of particular interest. Theseantibodies are identified by methods that are conventional per se. Forexample, a bank of candidate antibodies capable of binding to the bloodfactor are obtained by the above methods using immunization with thefull length polypeptide. These can then be subdivided by their abilityto bind to the various blood factor polypeptide domains usingconventional mapping techniques. Less preferably, antibodies specificfor a predetermined domain are initially raised by immunizing thesubject with a polypeptide comprising substantially only the domain inquestion, e.g. Factor X with an active serine protease domain. Theseantibodies may require routine mapping if binding to a particularepitope is desired.

Antibodies that are capable of binding to proteolytic processing sitesare of particular interest in the practice of this invention. They areproduced either by immunizing with a blood factor fragment that includesthe processing site or with intact blood factor and then screening forthe ability to block or inhibit proteolytic processing of the bloodfactor into the activated blood factor form. These antibodies are usefulfor suppressing the release of the activated blood factor and thereforeare promising for use in preventing the release of activated bloodfactor and stimulation of its pro- or anti-coagulant activities. Manysuch proteolytically active and proteolytic processing sites have beenmapped and are commonly known for the blood factors discussed herein. Asdescribed above, the antibodies should have high specificity andaffinity for the target sequence.

Isolated blood factors may be used in quantitative diagnostic assays asa standard or control against which samples containing unknownquantities of the blood factor may be compared.

Blood factor antibodies are useful in diagnostic assays for blood factorexpression in specific cells or tissues. The antibodies are labeled inthe same fashion as the blood factor described above and/or areimmobilized on an insoluble matrix.

Blood factor antibodies also are useful for the affinity purification ofthe blood factor from recombinant cell culture or natural sources. Bloodfactor antibodies that do not detectably cross-react with other bloodfactors, or which react only with the inactive, activated or inactivatedactive form of a particular blood factor can be used to purify thatblood factor free from other known ligands or other protein.

Suitable diagnostic assays for the blood factors of this invention andtheir antibodies are well known per se. Such assays include commonlyknown competitive and sandwich assays, and steric inhibition assays.Competitive and sandwich methods employ a phase-separation step as anintegral part of the method while steric inhibition assays are conductedin a single reaction mixture. Fundamentally, the same procedures areused for the assay of the blood factor and for substances that bind theblood factor, although certain methods will be favored depending uponthe molecular weight of the substance being assayed. Therefore, thesubstance to be tested is referred to herein as an analyte, irrespectiveof its status otherwise as an antigen or antibody, and proteins thatbind to the analyte are denominated binding partners, whether they beantibodies, cell surface receptors, or antigens.

Analytical methods for the blood factor of this invention or itsantibodies all use one or more of the following reagents: labeledanalyte analogue, immobilized analyte analogue, labeled binding partner,immobilized binding partner and steric conjugates. The labeled reagentsalso are known as "tracers."

The label used (and this is also useful to label blood factor encodingnucleic acid for use as a probe) is any detectable functionality thatdoes not interfere with the binding of analyte and its binding partner.Numerous labels are known for use in immunoassay, examples includingmoieties that may be detected directly, such as fluorochrome,chemiluminescent, and radioactive labels, as well as moieties, such asenzymes, that must be reacted or derivatized to be detected. Examples ofsuch labels include the radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I,technetium, fluorophores such as rare earth chelates or fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,luciferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, b-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No.3,645,090 (enzymes); Hunter et al., Nature, 144: 945 (1962); David etal., Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol.Methods, 40:219-230 (1981); and Nygren, J. Histochem. and Cytochem.,30:407-412 (1982). Preferred labels herein are enzymes such ashorseradish peroxidase and alkaline phosphatase. The conjugation of suchlabel, including the enzymes, to the antibody is a standard manipulativeprocedure for one of ordinary skill in immunoassay techniques. See, forexample, O'Sullivan et al., "Methods for the Preparation ofEnzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods inEnzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (AcademicPress, New York, N.Y, 1981), pp. 147-166. Such bonding methods aresuitable for use with the blood factor or its antibodies, all of whichare proteinaceous.

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the binding partner from any analytethat remains free in solution. This conventionally is accomplished byeither insolubilizing the binding partner or analyte analogue before theassay procedure, as by adsorption to a water-insoluble matrix or surface(Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling (forexample, using glutaraldehyde cross-linking), or by insolubilizing thepartner or analogue afterward, e.g., by immunoprecipitation.

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer analogue to competewith the test sample analyte for a limited number of binding sites on acommon binding partner. The binding partner generally is insolubilizedbefore or after the competition and then the tracer and analyte bound tothe binding partner are separated from the unbound tracer and analyte.This separation is accomplished by decanting (where the binding partnerwas preinsolubilized) or by centrifuging (where the binding partner wasprecipitated after the competitive reaction). The amount of test sampleanalyte is inversely proportional to the amount of bound tracer asmeasured by the amount of marker substance. Dose-response curves withknown amounts of analyte are prepared and compared with the test resultsto quantitatively determine the amount of analyte present in the testsample. These assays are called ELISA systems when enzymes are used asthe detectable markers.

Another species of competitive assay, called a "homogeneous" assay, doesnot require a phase separation. Here, a conjugate of an enzyme with theanalyte is prepared and used such that when anti-analyte binds to theanalyte the presence of the anti-analyte modifies the enzyme activity.In this case, the blood factor or its immunologically active fragmentsare conjugated with a bifunctional organic bridge to an enzyme such asperoxidase. Conjugates are selected for use with anti-blood factor sothat binding of the anti-blood factor antibody inhibits or potentiatesthe enzyme activity of the label. This method per se is widely practicedunder the name of EMIT.

Steric conjugates are used in steric hindrance methods for homogeneousassay. These conjugates are synthesized by covalently linking alow-molecular-weight hapten to a small analyte so that antibody tohapten substantially is unable to bind the conjugate at the same time asanti-analyte. Under this assay procedure the analyte present in the testsample will bind anti-analyte, thereby allowing anti-hapten to bind theconjugate, resulting in a change in the character of the conjugatehapten, e.g., a change in fluorescence when the hapten is a fluorophore.

Sandwich assays particularly are useful for the determination of bloodfactor or blood factor antibodies. In sequential sandwich assays animmobilized binding partner is used to adsorb test sample analyte, thetest sample is removed as by washing, the bound analyte is used toadsorb labeled binding partner, and bound material is then separatedfrom residual tracer. The amount of bound tracer is directlyproportional to test sample analyte. In "simultaneous" sandwich assaysthe test sample is not separated before adding the labeled bindingpartner. A sequential sandwich assay using an anti-blood factormonoclonal antibody as one antibody and a polyclonal anti-blood factorantibody as the other is useful in testing samples for blood factoractivity.

The foregoing are merely exemplary diagnostic assays for blood factorsand blood factor antibodies. Other methods now or hereafter developedfor the determination of these analytes are included within the scopehereof, including the bioassays described above.

All references cited in this specification are expressly incorporated byreference. The following examples are intended to illustrate, but notlimit the invention.

EXAMPLE 1 Construction of DNA Encoding Catalytically Inactive Forms ofRecombinant Human Factor X (rXi)

A full length cDNA clone for human Factor X was obtained from Dr. W. R.Church, University of Vermont (FIG. 4). This cDNA encodes the amino acidsequence of FIG. 1 or an allelic variant. This human Factor X cDNA wascloned into EcoRI site of vector pBSII (Stratagene) to obtain pBSX. TheHindIII-XbaI fragment of pBSX comprising the entire Factor X codingregion was subcloned into the Hind III-XbaI site of vector M13mplg(Mpl9X). Oligonucleotide site-directed mutagenesis was then performed asdescribed by Kunkel, T. A., et al., Methods in Enzymol (1987) 154:367.

The following forms were produced:

The oligomer TGC CGA GGG GAC GCC GGG GGC CCG CAC was used to convertserine (S₁₈₅) at position 185aa on the Factor X heavy chain to alanine(A₁₈₅) to obtain rXiA₁₈₅.

The oligomer ACC TAT GAC TTC AAC ATC GCC GTG CTC was used to convertaspartic acid (D₈₈) at position 88aa on the Factor X heavy chain toasparagine (N₈₈) to obtain the gene encoding rXiN₈₈.

Both oligomers were used to obtain the gene encoding rXiN₈₈ A₁₈₅. (SeeFIG. 1 for location of these sites). Verification ofoligonucleotide-directed mutagenesis was accomplished by dideoxysequencing.

EXAMPLE 2 Construction of DNA Encoding the Truncated Precursor of HumanFactor Xa (rX')

The cDNA of human Factor X (Mpl9X) was converted to encode varioustruncated forms of human Factor Xa, collectively designated rX', bydeletion of the activation peptide, by oligonucleotide site directedmutagenesis (Kunkel, T. A., et al., Methods in Enzymol (1987) 154:367).The following oligonucleotides employed with the corresponding aminoacid changes are as follows:

rX'Δ0: ACC CTG GAA CGC AGG AAG AGG ATC GTG GGA GGC CAG GAA TGC, whichaligned arginine (R142) following the C-terminus of the Factor X lightchain with isoleucine (I₅₃) 53aa of the Factor X activation peptide (1aaof the heavy chain);

rX'Δ1: ACC CTG GAA CGC AGG AAG AGG AGA ATC GTG GGA GGC CAG GAA TGC,which aligned this R₁₄₂ with arginine (R₅₂) of the Factor X activationpeptide;

rX'Δ2: ACC CTG GAA CGC AGG AAG AGG CGG AAA AGA ATC GTG GGA GGC CAG GAATGC, which extended R₁₄₂ following the Factor X light chain by two aminoacids arginine (R₁₄₃) and lysine (K₁₄₄) and aligned this terminus withR₅₂ of the Factor X activation peptide (FIG. 2);

rX'Δ3: ACC CTG GAA CGC AGG AAG AGG CCT AGG CCA TCT CGG AAA CGC AGG ATCGTG GGA GGC CAG GAA TGC, which extended R₁₄₂ following the Factor Xlight chain by seven amino acids, Proline (P₁₄₃) Arginine (R₁₄₄) Proline(P₁₄₅) Serine (S₁₄₆) Arginine (R₁₄₇) Lysine (K₁₄₈) Arginine (R₁₄₉) asdescribed in Ehrlich, H. J., et al. J Biol Chem (1989) 264:14298, andaligned this terminus with R₅₂ of the Factor X activation peptides.

Verification of the oligonucleotide directed mutagenesis wasaccomplished by dideoxy sequencing.

As will be further described below, the precursor derived from rXI Δ2was cleaved endogenously when recombinantly produced in CHO cells toobtain directly the activated form rXa. The precursor derived from rX'Δ0was not cleaved endogenously in CHO cells when produced recombinantly.The precursor derived from rX'Δ1 or from rX'Δ3 was cleaved incompletely.The dimeric peptides derived from rX'Δ0, rX'Δ1 and rX'Δ3 were not activeenzymatically.

EXAMPLE 3 Construction of DNA Encoding Catalytically Inactive TruncatedPrecursor (rX'i)

cDNA Factor X' constructs described in Example 2 were converted toencode the catalytically inactive forms of X' (rX'i) by oligonucleotidesite-directed mutagenesis as described in Example 1. These constructsincluded rX'i(Δ2)N₈₈ (rX'i(Δ2)N₈₈) and rX'i(Δ2)N₈₈ A₁₈₅, as shown inFIG. 3.

EXAMPLE 4 Expression of the Genes Encoding Precursor (rX and rX')

The expression vector pRC/CMV (Invitrogen) was modified by replacing theCMV promoter with the SRa promoter (Takabe, Y., et al., Molec Cell Biol(1988) 8:466). The ClaI-XbaI fragment, filled in by Klenow polymerase atthe ClaI site which contained the SRa promoter was isolated from theexpression vector pBJI (Lin, A., et al., Science (1990) 249:677 andavailable from M. Davis, Stanford University) and subcloned into theNruI-XbaI site of pRC/CMV creating expression vector pBN. The StuIfragment of pBN, comprising the SRa promoter, bovine growth hormonepolyadenylation site and M13 origin or replication was subcloned intothe StuI site of pSV2DHFR generating expression vector pBD. The Mp19SmaI-EcoRV fragments of the precursor DNAs described in examples weresubcloned into the Klenow polymerase filled-in XbaI site of pBN and pBD.The resulting expression vectors were transfected into CHO bylipofection (BRL). Selection for transfected clones was by either 1mg/ml G418 Neomycin (Gibco) or 25 ng/ml Methotrexate (Sigma). Singleclones were isolated by cloning cylinders, expanded and expressionlevels were determined on 24 hour serum free medium by a standard solidphase antibody capture assay (ELISA) as described by Harlow, E., andLane, D., in Antibodies (1988), Cold Spring Harbor Laboratory, New York.The ELISA utilized a primary antibody of rabbit polyclonal antihumanFactor X (STAGO, American Diagnostics Inc.) and a rabbit-specificsecondary antibody of peroxidase conjugated goat IgG.

Clones from constructs pBNX, pBNX'Δ0, pBNX'Δ1 pBNX'Δ2, and pBNX'Δ3 wereexpanded to confluence in T-7t tissue culture flasks in RPMI mediumsupplemented with 10% fetal bovine serum, Penicillin, Streptomycin,Glutamine and 10 μg/ml vitamin K, washed four times with serum freemedium and incubated overnight with serum free medium.

Post-incubation the medium was harvested, centrifuged at 3000 rpm and 2ml was precipitated with 10% Trichloracetic acid (TCA). The TCA pelletwas washed three times with 100% Acetone, resuspended to 0.05 mlSDS-PAGE sample buffer or 0.05 m SDS-PAGE sample buffer with 1M βMercaptoethanol. Duplicate 10 μl aliquots were electrophoresed on 12%SDS polyacrylamide gels and transferred to Immobilon filters(Millipore). Western blot analysis was performed with the primary humanFactor X polyclonal rabbit sera (STAGO, American Diagnostics, Inc.) at a1/4000 dilution in 1% nonfat dry milk, 0.1% NP40, 10 mM Tris-HCl pH 7.5,150 mm NaCl. The secondary antibody was ¹²⁵ I labeled Fab donkeyantirabbit IgG (Amersham). Autoradiography was overnight at -70° C. withan intensifier screen.

The pattern of antibody reactivity showed that the expected productswere produced. All five products, i.e., those derived from rX, rX'Δ0,rX'Δ1, rX'Δ2, and rX'Δ3 were positive in the above ELISA based on rabbitpolyvalent human Factor X antisera. ELISAs were also performed withrespect to mouse monoclonal antibodies Mab323, Mab743 and Mab325. Mab323is specifically reactive with the activation peptide. Mab743 is reactivewith either the activated or inactivated form of human Factor X. Mab325is calcium ion dependent and directed to the light chain; this antibodyreacts with the gammacarboxylated region.

Supernatants from cultures containing any of the five constructs gavepositive ELISAs with Mab743 and Mab325; thus, posttranslational GLAprocessing is indicated in all cases. All of the rX' mutants failed toreact with Mab323 confirming the absence of the activation peptide.

FIGS. 5a and 5b show Western blot analysis using polyclonal rabbitantisera of products derived from rX, rX'Δ0, rX'Δ1, rX'Δ2, rX'Δ3 and CHOcontrol medium. Rabbit polyclonal antisera to X was not efficient inlocalizing the fully processed heavy chain of human Factor Xa; hence, inall cases the position expected to be occupied by the activated heavychain does not appear. FIG. 5a shows reduced and FIG. 5b nonreducedforms of these recombinant proteins. Lane 1, 0.7 ,mg native human FactorX (Dr. C. Esmon, OMRF, University of Oklahoma); Lane 2, rX; Lane 3,rX'Δ0; Lane 4, rX'Δ1; Lane 5 rX'Δ2 Lane 6, rX'Δ3; Lane 7, CHO controlmedium.

FIG. 5a shows that the recombinant products of rX and rX'Δ2 are dimericproteins which are separable under reducing conditions. The products ofexpression of rX'Δ0, rX'Δ1 and rX'Δ3 apparently are largely single chainproducts. It appeared that the unprocessed Factor X' single chainscomigrated anomalously with the heavy chain as shown in lanes 3-7,apparently due to the degree of proteolytic processing of the novelcleavage sites. The failure of these X' precursor proteins to beprocessed properly was consistent with the results of a coagulationassay, described in Example 8, which demonstrated that Factor Xa,RVV-activated Factor X or recombinant Factor X and X1Δ2 were comparablyactive, while the remaining X' secreted products were dramatically lessefficient, by at least 5 or magnitude. The data with respect to enzymeactivity are shown in Table 1:

                  TABLE 1                                                         ______________________________________                                        Factor X                                                                             RVV Activation                                                                            Catalytic Efficiency (%)                                                                     Coagulation                                 ______________________________________                                        X      +           100            +                                           XA                               851                                                                                                 +                      RX                               29.6                                                                                               +                       X'.increment.0                                                                                                 5.2 × 10.sup.-4                                                            -                                         X'.increment.1                                                                                                 12.6 × 10.sup.-4                                                          -                                          X'.increment.2                                                                                                 269                                                                                                 -                      X'.increment.3                                                                                                 69.5 × 10.sup.-4                                                          -                                          CHO                               0                                           ______________________________________                                                                          -                                       

The column of Table 1 labeled "catalytic efficiency" shows theamidolytic substrate activities of the various factors, activated withRVV if necessary. The catalytic efficiencies shown are the ratio ofkcat/Km and were normalized to the results for plasma Factor X. As shownin the table, both recombinant Factor X and X'Δ2 were active in Factor Xdependent 2PT clotting assays, while the enzymatic activities of theother recombinant proteins were 4 orders of magnitude lower.

From FIG. 5b, it is apparent that the expression products of theX'-encoding gene are of lower molecular weight than rX or native FactorX.

EXAMPLE 5 Purification of rX and X'Δ2

Both recombinant Factor X and X'Δ2 were purified to homogeneity asfollows: After growth to confluence, CHO cells transfected with pBNX orpBNX'Δ2 were washed 4-5 times with serum-free media. The cells were thencultured for consecutive 24 hr periods at 37° C. in serum-free mediasupplemented with 4 μg/ml vitamin K3.

Harvested media were centrifuged at 15,000× g for 20 min followed byfiltration of the supernatant through a 0.2 μm filter. To the media wasadded Tris HCl, pH 7.5 to 20 mM, NaEDTA to 10 mM, and the resultant waschromatographed on Q-Sepharose Fast Flow (Pharmacia). Allchromatographic steps were performed at 4° C. The columns were washedextensively with 20 mM Tris, pH 7.5, 10 mM EDTA, 0.15 M NaCl, and theproteins were eluted with 20 mM Tris, pH 7.5, 0.5M NaCl, 5 mM CaCl₂.Peak fractions were pooled and either stored frozen at -20° C. orapplied directly to an anti-factor X monoclonal antibody affinity columnas described by Church, W. R., et al., Throm Res (1985) 38:417-424. Theantibody used for isolation (aHFX-Id, Mab B12-A3) is specific for humanfactor rX, not influenced by Ca² +, and binds both factors X and Xa(unpublished data). Factor rX' was purified further on abenzamidine-Sepharose column (Pierce) as described by Krishnaswamy, etal., J Biol Chem (1987) 262:3291-3299. The concentrations of theproteins were determined by quantitative ELISA, colorimetric proteinassay (Harlow, E., et al., "Antibodies, A Laboratory Manual" (1988),Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.), and byabsorbance measurement at 280 nm using extinction coefficient 11.6 andmolecular weights of 58,900 for factor X, 46,000 for X'Δ2.

The purified factors, when subjected to SDS-PAGE under reducingconditions and silver stained showed that the recombinantly producedFactor X was separated into 3 bands representing the full-lengthprecursor (75 kD), the heavy chain containing the activation peptide (45kD) and the light chain (22 kD). When amino terminal sequence analysiswas performed following electrotransfer to nylon filters, the lightchain was shown to be heterogeneous with 27% initiating at Val₃₇ and 73%initiating at Ala₄₁ ; the 75 kD species was also heterogeneous with 41%initiating at Val₃₇ and 59% initiating at Ala₄₁.

EXAMPLE 6 Expression of Genes Encoding Inactivated Recombinant HumanFactor X (rXi and rX'i)

The X' form chosen for conversion to the inactive form was the rX'Δ2form shown in FIG. 4. pBN-derived cell lines for rX, rX' ('2), rXiN₈₈A₁₈₅, rXiA₁₈₅, rX'i(Δ2)N₈₈ A₁₈₅ and rX'iN₈₈ (Δ2) were grown toconfluence in 800 cm² roller bottles as described in Example 4, washedfour times with serum free medium and incubated overnight with 50 mlserum-free medium. The medium was replenished and harvested daily.

Consecutive harvests were pooled, centrifuged at 3000 rpm and passeddirectly through a Factor X-specific monoclonal antibody (Mab) affinitycolumn (Mab717) supplied by Dr. C. Esmon (OMRF, University of Oklahoma).The bound "Factor X1" was eluted from the Mab717 column with 80%ethylene glycol, dialyzed against 10 mM Tris HCl, pH 7.5, 150 mM NaCland concentrated on a Centricon 10 filtration unit (Amicon). "Factor X"protein concentrations were determined by ELISA as described in Example4 utilizing serial dilution with comparison to a standard preparation ofhuman Factor X (Haematologic Technologies, Inc., C. Esmon, OMRF,University of Oklahoma).

The purified proteins were characterized by Western blot analysis asoutlined in Example 4. FIG. 6 shows a Western blot of these,b-mercaptoethanol-reduced, Mab717 purified recombinant human Factor Xanalogs. Lane 1, 0.1 mg human X (Haematologic Technologies, Inc.); Lane2, 0.1 mg human Xa (Haematologic Technologies, Inc.); Lane 3, 0.1, mgrX; Lane 4, 0.16 mg rX'Δ2; Lane 5, 0.13 mg rXiN₈₈ A_(185;) Lane 6, 0.15mg rXiA₁₈₅ ; Lane 7, 0.187 mg rX'i(Δ2)N₈₈, Lane 8, 0.05 mg rX'i(Δ2)N₈₈A₁₈₅.

It is evident that, under reducing conditions, human X and human Xa arein dimeric form; human Xa shows a lower molecular weight form of theheavy chain due to the absence of the activation peptide. Recombinanthuman X in lane 3 is similar to native human X, however some singlechain precursor is still evident. In lane 4, recombinant rX'Δ2 alsoshows cleavage to the heavy and light chains. In lanes 5 and 6, themodified recombinant X1 proteins behave in a manner similar torecombinant human X. As expected, lanes 7 and 8 show the presence ofmonomer, heavy and light chains derived from the proteolytic cleavage ofX'i.

EXAMPLE 7 Enzymatic Analysis of Recombinant Human Factor X

The kinetic measurement of chromozym X(N-methoxycarbonyl-D-norleucyl-glycyl-arginine-4-nitranilide acetate,Boehringer Mannheim) hydrolysis by native human Factor X, Xa,recombinant X (rX), rX1Δ0, rX1Δ1, rX1Δ2, rX1Δ3, rXiN₈₈ A₁₈₅, rXiN₈₈,rX'(Δ2N₈₈ A₁₈₅ and inactivated bovine Xa, Xai-APMSF supplied by Dr. C.Esmon (OMRF, University of Oklahoma) (Skogen, W. F., et al, J Biol Chem(1984) 259:2306) were examined at room temperature in 96-well microtiterplates on a Molecular Devices Vmax spectrophotometer. The absorbance at405 nM was monitored continuously and the reaction velocities weredetermined directly by the machine and plotted with the Enzfitterprogram (Elsevier Press). Protein concentrations were determined byELISA (Example 6). All enzymes were diluted to the appropriateconcentration in 0.1% bovine serum albumin (BSA) 50 mM Tris HCl, pH 8.0,150 mM NaCl. Duplicate reactions were carried out in 50 mM Tris HCl, pH8.0, 150 mM NaCl and 2.5 mM CaC12. All recombinant human Factor X's wereMab717-purified (Example 6) except for RX'D0, RX'D1, and RX'D3 whichwere purified using QAE-Sepharose (Pharmacia) concentrated (Skogen, W.F., et al., J Biol Chem (1984) 259:2306).

The recombinantly produced peptides derived from vectors containing rX,rXiN₈₈ A₁₈₅, rXiN₈₈ and rXaiN₈₈ A₁₈₅ were treated by preincubation for 5minutes with Russell's viper venom to convert them to the Xa or Xaiform. Peptides derived from the rX'Δ0, rX'Δ1, rX'Δ2 and rX'Δ3 vectorswere not treated in this fashion.

FIG. 7 is a comparison of Lineweaver-Burk plots for native human FactorX and Xa and activated forms derived from recombinant human rX and rX'.FIG. 7a, human X; FIG. 7b, human Xa; FIG. 7c, human rX (treated withRussell's viper venom protease); FIG. 7d, human rX' (not treated withprotease).

Table 2 compares the Kcat and Km values of the recombinantly producedhuman Factor X's to the native human Factor X and Xa supplied byHaematologic Technologies, Inc.

                  TABLE 2                                                         ______________________________________                                                             Specificity Constant                                               Kcat (s.sup.-1)                                                                       Km (mm)  Kcat/Km (s.sup.-1 M.sup.-1)                        ______________________________________                                        Native Forms                                                                  X                                     489 × 10.sup.3                    Xa                                1996 × 10.sup.3                       Precursor construct                                                           rX                                    167 × 10.sup.3                    rX'.increment.1                                                                                    N.D.                                                                                                --                                 rX'.increment.2                                                                                    N.D.                                                                                                --                                 rX'.increment.3                                                                                    17                                                                                             115 × 10.sup.3                    rXiN.sub.88 Aa.sub.185                                                                    N.D.                           --                                 rXiA.sub.185                                                                                       N.D.                                                                                                --                                 rX'iN.sub.88 A.sub.185                                                                    N.D.              --                                                                                         --                                 rX'iN.sub.88                                                                                    N.D.                                                                                                   --                                 Control CHO medium                                                                          N.D.                         --                                 ______________________________________                                         N.D. = not detected, Kcat ≦ .1 in 14 hrs-16 hrs assay.            

Of course, none of the inactivated forms give values; of the rX' forms,only rX'Δ2 showed activity.

EXAMPLE 8 Factor X Dependent Prothrombinase Complex Activity of Human X,Xa and Recombinant Human rX and rX'

Factor X dependent prothrombinase complex activity was determined bymeasuring the rate of chromozyme TH (tosyl-glycyl-prolyl-arginine-4nitroanilide acetate, Boehringer Mannheim) hydrolysis by thrombin atroom temperature in a 96-well microtiter plate on a Molecular DevicesVmax spectrophotometer. The absorbance at 405 nM was continuouslymonitored and the initial one minute reaction velocities were determineddirectly by the machine and plotted using the Enzfitter program(Elsevier). Reaction mixtures were performed in triplicate with0.05×10⁻⁴ M to 1.5×10⁻⁹ M "Factor X," determined by ELISA (Example 6),0.5×10⁻⁶ M human prothrombin (STAGO, American Diagnostics, Inc.)7.5×10⁻⁹ M human factor Va (Haematologic Technologies, Inc.), 20×10⁻⁶ Mphosphocholine/phosphoserine 75%/25% (PCPS) (supplied by Dr. W. R.Church, University of Vermont), or equivalent amounts of rabbit braincephalin (Sigma) (Example 9), 0.1% BSA (Sigma), 0.1×10⁻³ M chromozym TH(Boehringer Mannheim), 25 mM Tris HCl, pH 7.5, 150 mM NaCl and 5 mMCaC12.

Human Factor rX and rX' dependent prothrombinase complex activityutilized PCPS and human Factor X and Xa dependent prothrombinase complexactivity utilized cephalin. Human Factor X and rX were preincubated for5 minutes with Russell's viper venom (Haematologic Technologies, Inc.).Thrombin hydrolysis of chromozym TH as determined by increase offluorescence signal, was linear throughout the experimental protocol. Noobservable rates were shown for rXiN₈₈ A185 at 59.2×10⁻⁴ M, rX'iN₈₈ A₁₈₅at 10.2×10⁻⁹ M, or for bXaiAPMSF at 1×10⁻⁹ M. FIGS. 8a-8d compare FactorX dependent prothrombinase complex activity of human X (FIG. 8a), humanXa (FIG. 8b) (Haematologic Technologies, Inc.), recombinant human rX(after treatment with protease) (FIG. 8c) and recombinant human rX'Δ2(after no protease treatment) (FIG. 8d). All are comparably active.

EXAMPLE 9 Coagulation of Plasma

Mab717 purified rX and rXa were assayed for plasma coagulation activityin an automated two-stage prothrombin assay on a MLA Electra 800fibrometer. Enzyme protein concentrations were determined by ELISA(Example 6) and diluted in 0.1% BSA, 150 mM NaCl prior to use. BovineFactor X and Factor VII deficient plasma (Sigma) and rabbit braincephalin (sigma) were prepared according to manufacturers' instructions.Russell's viper venom 0.1 ,μg/ml was added to human X and rX assays. Thereaction mixture comprised 0.1 ml Factor X, 0.1 ml 150 mM NaCl, 0.1 mlcephalin and 0.1 ml 25 mM CaCl2. Duplicates were performed on eachconcentration and the average of two experiments were calculated. FIG. 9compares the plasma coagulation activity of human X, human Xa, human rXand human rXa. Human rX was calculated to be 45% as active as human Xand human rXa was calculated to be 32% as active as human Xa.

EXAMPLE 10 Inhibition of Prothrombinase Complex Activity by rXiN₈₈ A₁₈₅.Human rX'i)Δ2)N₈₈ A₁₈₅ and Bovine bXai-APMSF

Inhibition of native human Factor X dependent prothrombinase complexactivity by human rXiN₈₈ A₁₈₅ and inhibition of native human Factor5×10⁻⁹ M Xa dependent prothrombinase complex activity by human rXi(Δ2)N₈₈ A₁₈₅ (rXai) and bovine bXai-APMSF (C. Esmon, OMRF, University ofOklahoma) was tested as detailed in Example 8. It is necessary tocompare directly X with Xi and Xa with Xai because of kinetic factorsand the strength of the complex once formed. Human rXiN₈₈ A₈₁₅ waspreincubated for 5 minutes with 0.1 mg/ml Russell's viper venom. Thehuman Factor X and Xa concentrations were 1×10⁹ M.

FIG. 10 shows the concentration dependent inhibition of the human FactorXa dependent prothrombinase complex by bXai-APMSF, rX'i(Δ2)N₈₈ A₁₈₅(rXai) and inhibition of the human Factor X dependent prothrombinasecomplex by rXiN₈₈ A₁₈₅. 50% inhibition by bXai-APMSF was obtained at0.9×10⁻⁹ M, 50% inhibition by rX'i(Δ2)N₈₈ A₁₈₅ was obtained at 6×10⁻⁹ Mand 50% inhibition by rXiN₈₈ A₁₈₅ was obtained at 10.6×10⁻⁹ M.

EXAMPLE 11 Preparation of Acylated Factor Xa

Human factor Xa was prepared as described above, and treated with a 3fold molar excess of p-amidinophenyl p'-anisate or p-amidinophenylp'-toluate. At different time points, an aliquot was removed from thereaction mixture and assayed for factor Xa dependent amidolyticactivity. The plot in FIG. 11 depicts residual amidolytic activityversus time of chemical modification.

Next, evaluations were made of the activation of the acyl Xa. Acylatedfactor Xa was incubated in a buffer at pH 7.5 at room temperature or 37°C. Over the time course of experimentation, withdrawn aliquots wereassayed for factor Xa activity. A sample of unmodified human factor Xawas subjected to the same incubation protocol. FIGS. 12a and 12b depictthe relative percent activity of incubated acyl Xa.

EXAMPLE 12 Clotting Activities of acyl-Xa

Acylated inactive human Factor Xa (acyl-Xa) was prepared as describedabove, and its in vitro and in vivo properties were studied. It wasfound that acyl-Xa deacylates and regains factor Xa catalytic activityin a time-dependent manner. Recovery rates were influenced by thestructure of the acyl-leaving group, temperature and pH. DeacylatedFactor Xa demonstrated normal amidolytic, prothrombinase and plasmaclotting activities in vitro. In vivo bolus administration of anisoyl-Xa(p-amidinophenyl p'anisate) at 2, 10, and 50 μg/kg in normal rabbits,dogs and hemophilic dogs demonstrated dose-dependent procoagulentactivity as measured by APTT and PT clotting times (data not shown).Procoagulant activity was time-dependent and correlated with circulatingplasma levels. No significant changes in other hemostatic parameterswere observed (TCT, Fibrinogen, FVIII, FIX, CBC).

The effects of infusion of p-anisoyl Xa in rabbits was also studied.Acyl Xa was infused into anesthetized New Zealand rabbits. Infusioncontinued for two hours and blood samples were collected from thefemoral vein at different time points during this period. FIG. 13depicts ex-vivo clotting (APTT) over the time course of infusion. Theratios are expressed relative to pre drug control.

EXAMPLE 13 Activation of other acylated proteins

Activation of acyl activated protein C is shown in FIG. 14. Acylated aPCprepared as described above was incubated in a buffer at pH 7.5 at roomtemperature. Over the time course of experimentation, withdrawn aliquotsare assayed for aPC activity in a chromogenic assay. A sample ofunmodified human aPC was subjected to the same incubation protocol. FIG.14 depicts the relative percent activity in incubated acyl aPC versuscontrol aPC.

Activation of o-anisoyl factor VIIa is shown in FIG. 15. Acylated factorVIIa was prepared as described above and incubated in buffer at aprotein concentration of 160 nM. At each time point an aliquot wasdiluted to 0.16 nM and incubated with lipidated tissue factor (0.25 nM)for one minute at room temperature. The factor VIIa/Tissue Factormixture was then used for activation of factor X and the resultingfactor Xa was assayed in an amidolytic assay. Results are shown in FIG.15.

We claim:
 1. An indwelling intravascular device comprising aprocoagulant composition comprising Factor Xa and biologically activepolypeptide fragments thereof, which Factor Xa and fragments thereofhave been transiently inactivated to have little or no enzymaticactivity, and wherein said inactivated Factor Xa and fragments thereofgenerate an active form of Factor Xa in the presence of serum.
 2. Theindwelling intravascular device of claim 1, wherein the Factor Xa orfragments thereof comprises a consecutive sequence of at least 5, 10,15, 20, 25, 30 or 40 amino acids of Factor Xa.
 3. The indwellingintravascular device of claim 2, wherein said device is selected fromthe group consisting of i.v.s, patch, tubing, vascular stent, catheterand vascular graft.
 4. The indwelling intravascular device of claim 2,wherein the Factor Xa or fragments thereof are transiently inactivatedby an antibody or antibody fragment thereof.
 5. The indwellingintravascular device of claim 4, wherein said antibody is a monoclonalantibody.
 6. The indwelling intravascular device of claim 4, wherein theantibody fragment is a Fab fragment.
 7. The indwelling intravasculardevice of claim 2, wherein the Factor Xa or fragments thereof aretransiently inactivated by an inhibitor selected from the groupconsisting of benzamidines, substituted benzamidines and Kunitz classinhibitors.
 8. The indwelling intravascular device of claim 7, whereinthe inhibitor is a Kunitz class inhibitor and is further selected fromthe group consisting of aprotinin and LACI.
 9. The indwellingintravascular device of claim 2, wherein the Factor Xa and fragmentsthereof are transiently inactivated by 1,2-bis(5-amidino2-benzofuranyl)ethane.
 10. The indwelling intravascular device of claim2, wherein the Factor Xa and fragments thereof are transientlyinactivated by an acyl group.
 11. The indwelling intravascular device ofclaim 10, wherein the Factor Xa and fragments thereof are transientlyinactivated by an acyl group selected from the group consisting ofunsubstituted and substituted benzoyl, unsubstituted and substitutedacryloyl with the proviso that the substituted acryloyl is not a phenylsubstituted acryloyl, unsubstituted and substituted acetyl,unsubstituted and substituted carbonyl, and unsubstituted andsubstituted isocoumarins.
 12. The indwelling intravascular device ofclaim 10, wherein the acyl group is an unsubstituted or substitutedbenzoyl selected from the group consisting of benzoyl, p-fluoro benzoyl,o-fluoro benzoyl, p-alkoxy benzoyl, o-alkoxy benzoyl, 4-amino benzoyl,4N,N-dimethylamino benzoyl,4N-(2-N'-(3-(2-pyridyldithio)-propenyl)amino-ethyl)amino benzoyl,guanidino benzoyl, 4-amidinophenyl-p-anisoyl, p-anisoyl, o-anisoyl,4-amidinophenyl-p-toluoyl, p-toluoyl and o-toluoyl.
 13. The indwellingintravascular device of claim 10, wherein the acyl group is anunsubstituted or non-phenyl substituted acryloyl selected from the groupconsisting of 3,3-dimethyl acryloyl, 3,4-dimethyl acryloyl, 3,3-difluoroacryloyl and 3,4-difluoro acryloyl.
 14. The indwelling intravasculardevice of claim 10, wherein the acyl group is an unsubstituted orsubstituted acetyl selected from the group consisting of cyclohexylidineacetyl, 1-methylcyclohexylidine acetyl, methyl phenyl ketone andacetanilide.
 15. The indwelling intravascular device of claim 10,wherein the acyl group is an unsubstituted carbonyl of cyclohex-1-enecarbonyl.
 16. The indwelling intravascular device of claim 10, whereinthe acyl group is an unsubstituted or substituted isocoumarin selectedfrom the group consisting of 3-alkoxy 4-chloroisocoumarins.